Abstract:

The present invention is concerned with cancer treatment and diagnosis,
especially with melanoma associated peptide analogues with improved
immunogenicity, epitopes thereof, vaccines against melanoma, tumor
infiltrating T lymphocytes recognizing the antigen and diagnostics for
the detection of melanoma and for the monitoring of vaccination. The
peptides according to the invention can be exploited to elicit native
epitope-reactive Cm. Usage of the peptides with improved immunogenicity
may contribute to the development of CTL-epitope based vaccines in viral
disease and cancer.

33. Tumor infiltrating lymphocytes (TIL) which bind to the peptide of
claim 17.

34. Tumor infiltrating lymphocytes (TIL) which bind to the peptide of
claim 18.

35. An antibody directed against the peptide of claim 17.

36. An antibody directed against the peptide of claim 18.

37. A method of eliciting a T-cell response in a mammal comprising
administering to the mammal the peptide of claim 17 in an amount
sufficient to elicit a T-cell response in a mammal.

38. A method of eliciting a T-cell response in a mammal comprising
administering to the mammal the peptide of claim 18 in an amount
sufficient to elicit a T-cell response in a mammal.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of application Ser. No.
09/214,836 filed Oct. 4, 1999, which was the National Stage of
International Application No. PCT/EP97/03712 filed Jul. 8, 1997 and
having a priority date of Jul. 11, 1996. The disclosure of each of these
related applications is incorporated herein in their entireties.

BACKGROUND OF THE INVENTION

[0002]The present invention is concerned with cancer treatment and
diagnosis, especially with melanoma associated peptide analogues,
epitopes thereof, vaccines against and diagnostics for the detection of
melanoma and for the monitoring of vaccination.

[0003]During the stepwise changes from normal to tumor tissue,
tumor-associated antigens appear. The characteristics of tumor-associated
antigens are very much dependent on the origin of the tumor carrying
them. The existence of antigens associated with animal tumors was
documented in the last century, and the antigenic character of human
cancers has been well established, primarily through recent studies with
monoclonal antibodies.

[0004]Attempts to isolate and chemically characterize these antigens have
encountered serious difficulties, many having to do with a lack of
reagents suitable for precipitation of the antigen-bearing molecules from
a solution.

[0005]Like many other stimuli, the tumor-associated antigens activate not
one but a whole set of defense mechanisms--both specific and unspecific,
humoral and cellular. The dominant role in in vivo resistance to tumor
growth is played by T lymphocytes. These cells recognize tumor-associated
antigens presented to them by antigen presenting cells (APCs), and will
be activated by this recognition, and upon activation and
differentiation, attack and kill the tumor cells.

[0006]Cytotoxic T lymphocytes (CTL) recognize short peptide fragments of
9-11 amino acids in length, which are presented in the antigen-binding
groove of Major Histocompatibility Complex (MHC) class I molecules
(Townsend et al., 1986, Cell 44.959; Bjorkman et al., 1987, Nature
329:512). These peptides are usually derived from intracellular protein
pools and associate in the lumen of the endoplasmic reticulum with MHC
class I heavy chain and β2-microglobulin molecules, followed by
transportation of the MHC-peptide complex to the cell surface. Despite
the presence of many putative antigenic peptides within the same antigen,
only a few peptides are selected for recognition by CTL.

[0007]MHC Class I/II antigens are often down regulated in solid tumors.
This may affect all class I/II antigens, or only part of them. Viral and
cellular peptides that can sensitize appropriate target cells for
cytotoxic T lymphocyte mediated lysis may fail to do so when produced in
cells with a low level of expression of MHC class I antigen. Cytotoxic
sensitivity may be induced, at least in some cases by raising the level
of MHC class I/II antigen expression by interferon γ and tumor
necrosis factor α.

[0008]The MHC class I binding-affinity of an epitope is an important
parameter determining the immunogenicity of the peptide-MHC complex.
Analysis of Human histocompatibility antigen (HLA-A *0201)-restricted
epitopes recognized by anti-viral CTL demonstrated that several peptides
bind to HLA-A *0201 with high affinity. Furthermore, immunogenicity
analysis of motif containing potential epitopes using HLA-A *0201
transgenic mice revealed that a threshold MHC class I affinity was
required for a peptide in order to elicit a CTL response Messing et al.,
1995, J. Immunol. 154:5934; Sette et al., 1994, J. Immunol. 153:5586). In
addition to the MHC class I-binding affinity, stability of peptide-MHC
complexes at the cell surface contributes to the immunogenicity of a CTL
epitope. Consequently, MHC class I binding-affinity and stability of
peptide-MHC complexes are important criteria in the selection of specific
peptide determinants for development of CTL-epitope based therapeutic
vaccines.

[0012]In an attempt to improve the immunogenicity of two HLA-A *0201
presented epitopes derived from the melanocyte differentiation antigens
gp 100 and Melan-A/MART-1, amino acid substitutions within the epitopes
to improve HLA-A *0201-binding affinity were performed.

[0013]Surprisingly, it was found that these epitope-analogues have an
improved immunogenicity in view of the original epitope. Furthermore, in
the present invention it is demonstrated that the epitope-analogues allow
the induction of peptide-specific CTL displaying cross-reactivity with
target cells endogenously processing and presenting the native epitope.

[0014]Usage of these epitope-analogues according to the present invention
with improved immunogenicity may contribute to the development of
CTL-epitope based vaccines in chronic viral disease and cancer.

[0015]In more detail, since MHC class I-affinity and peptide-MHC
complex-stability are important parameters determining the immunogenicity
of an MHC class I presented epitope, the possibility to improve the
capacity of two melanocyte differentiation antigen-derived epitopes to
bind to HLA-A *0201 without affecting interactions with the T-cell
receptor (TCR) is explored. Detailed analysis of the Melan-A/MART-1 27-35
and gp100 154-162 epitopes using alanine substitutions revealed that
amino acids at positions 4 to 7 (Melan-A/MART-1 27-35) or 5 to 7 (gp100
154-162) are critical residues for TCR recognition. These data are in
line with X-ray crystallography studies of the HLA-A *0201 molecule
(Saper et al., 1991, J. Mol. Biol. 219:277; Latron et al., 1992, Science
257:964), implying a role for the more permissive residues at position 4
and 5 of the peptide oriented towards the outside of the MHC molecule, as
prominent TCR contact sites. It is demonstrated that for HLA-A *0201 the
amino acids at positions 6 and 7 of the Melan-A/MART-1 27-35 and gp100
154-162 epitopes do not only interact with secondary pockets in the MHC
peptide-binding cleft, but that they are also critical residues for TCR
interaction (Ruppert et al., 1993, Cell 74:929; Madden et al., 1993, Cell
75:693).

[0016]Surprisingly, the alanine substitution at position 8 in the gp100
154-162 epitope, KTWGQYWAV (SEQ ID NO: 1), resulted in a peptide that
displayed increased HLA-A *0201 affinity. Moreover, this epitope-analogue
was recognized by gp100-reactive CTL at tenfold lower concentrations
compared to the native epitope. These data demonstrate that amino acid
substitutions at a non-anchor position can result in increased MHC class
I affinity and T cell recognition.

[0017]By N-terminal anchor replacements with V, L, M or I towards the
HLA-A *0201 binding-motifs were set out to identify epitope-analogues for
both epitopes with improved affinity for HLA-A *0201 that were still
recognized by wild type epitope-reactive CTL. For the Melan-A/MART-1
epitope, epitope-analogues were obtained with comparable (M) or improved
(V, L and I) affinity for HLA-A *0201. However, all N-terminal anchor
replacements resulted in decreased T cell reactivity. Apparently, in case
of this epitope, the N-terminal anchoring residue affects the positioning
of the side chains in the center of the peptide, thereby abrogating TCR
interactions. Recently, a similar observation has been described
involving an HLA-B*3501 restricted epitope of the influenza A matrix
protein (Dong et al., 1-996, Eur. J. Immunol. 26:335). Substitution of a
serine residue at position 2 of the peptide for the more common
HLA-B*3501 N-terminal anchor proline, considerably enhanced binding to
HLA-B*3501, but the epitope-analogue was not recognized by CTL reactive
with the native epitope. Moreover, this peptide behaved as a
peptide-antagonist as was demonstrated for T cell recognition of both MHC
class II and class I-presented peptides (Dong et al., 1996, Eur. J.
Immunol. 26:335; De Magistris et al., 1992, Cell 68:625; Klenerman et
al., 1994, Nature 369:403). These findings illustrate that anchor residue
substitutions not only affect MHC class I binding, but in some cases they
may also result in a conformational change of the peptide-MHC complex,
leading to an altered interaction with the TCR.

[0018]However, in case of the gp100 154-162 epitope, in addition to the
alanine substituted analogue KTWGQYWAV (SEQ ID NO: 1), three anchor
substituted epitope-analogues KVWGQYWQV (SEQ ID NO: 2), KLWGQYWQV (SEQ ID
NO: 3), and KIWGQYWQV (SEQ ID NO: 4), with improved HLA-A*0201-affinity
that were recognized by anti-gp100 CTL at tenfold lower concentrations
compared to the wild type epitope were obtained. In vivo immunization
experiments using HLA-A*0201/Kb transgenic mice demonstrated that
these epitope-analogues were immunogenic, resulting in the induction of
murine CTL reactive with both the epitope-analogues and the native
epitope. The immunogenicity of the epitope-analogues was expected since
the peptide-MHC complex stability of both the epitope-analogues and the
native epitope was comparably high.

[0019]In vitro CTL induction experiments using donor derived PBL
demonstrated that epitope-analogue specific CTL could be obtained
displaying cross-reactivity with tumor cells endogenously presenting the
wild type epitope. In addition to T lymphocytes reactive with the wild
type epitope, the T cell repertoire of healthy donors apparently also
contains T cells reactive with the gp100 154-162 epitope-analogues.
Analysis of TCR usage of cloned CTL reactive with the different gp100
154-162 epitope-analogues and with wild type gp100 154-162 will be
informative of the spectrum of the T cell repertoire that can be used to
induce CTL reactivity towards the wild type epitope. With respect to
immunotherapy of cancer, activation of multiple specificities in the T
cell repertoire against an antigenic tumor epitope using
epitope-analogues may increase the possibility of a patient to mount a
successful anti-tumor response upon immunization. In addition, modified
epitopes might still elicit immune responses if tolerance against the
wild-type epitope is observed.

[0020]Employment of "improved" epitopes in immunotherapy protocols
increases the amount of peptide-MHC complexes at the cell surface of
antigen presenting cells in vivo, and will result in enhanced priming of
antigen-specific CTL. Apart from their potential ill cancer
immunotherapy, usage of epitope-analogues with improved immunogenicity
may contribute to the development of CTL-epitope based vaccines in
chronic viral disease.

[0021]Therefore, the present invention includes peptides, immunogenic with
lymphocytes directed against metastatic melanomas, characterized in that
it comprises at least part of the amino-acid sequence of SEQ ID NO: 9
wherein the amino-acid at position 2 or 8 is substituted.

[0022]A preferred embodiment of the present invention are peptides,
wherein at position 2 Threonine is substituted by Isoleucine, Leucine or
Valine.

[0023]Another preferred embodiment of the present invention are peptides,
wherein at position 8 Glutamine is substituted by Alanine.

[0024]A specific preferred embodiment of the present invention are
peptides, characterized in that it comprises the amino-acid sequence of
any of SEQ ID NOS: 1-4 or 32-34.

[0025]The term "peptide" refers to a molecular chain of amino acids, does
not refer to a specific length of the product and if required can be
modified in vivo or in vitro, for example by manosylation, glycosylation,
amidation, carboxylation or phosphorylation: thus inter alia
polypeptides, oligopeptides and proteins are included within the
definition of peptide. In addition, peptides can be part of a (chimeric)
protein or can be (part of) an RNA or DNA sequence encoding the peptide
or protein.

[0026]Of course, functional derivatives as well as fragments of the
peptide according to the invention are also included in the present
invention. Functional derivatives are meant to include peptides which
differ in one or more amino acids in the overall sequence, which have
deletions, substitutions, inversions or additions. Amino acid
substitutions which can be expected not to essentially alter biological
and immunological activities have been described. Amino acid replacements
between related amino acids or replacements which have occurred
frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly,
Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and
structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,
suppl. 3). Based on this information, Lipman and Pearson developed a
method for rapid and sensitive protein comparison (Science 227,
1435-1441, 1985) and determining the functional similarity between
homologous polypeptides.

[0027]Furthermore, as functional derivatives of these peptides are also
meant to include other peptide-analogues derived from gp100 (or Melan)
that are able to induce target cell lysis by tumor infiltrating
lymphocytes.

[0028]In addition, with functional derivatives of these peptides are also
meant addition salts of the peptides, amides of the peptides and
specifically the C-terminal amides, esters and specifically the
C-terminal esters and N-acyl derivatives specifically N-terminal acyl
derivatives and N-acetyl derivatives.

[0029]The peptides according to the invention can be produced
synthetically, by recombinant DNA technology or by viruses, if the amino
acid sequence of the peptide is encoded by a DNA sequence which is part
of the virus DNA. Methods for producing synthetic peptides are well known
in the art.

[0030]The organic chemical methods for peptide synthesis are considered to
include the coupling of the required amino acids by means of a
condensation reaction, either in homogenous phase or with the aid of a
so-called solid phase. The condensation reaction can be carried out as
follows:

[0031]condensation of a compound (amino acid, peptide) with a free
carboxyl group and protected other reactive groups with a compound (amino
acid, peptide) with a free amino group and protected other reactive
groups, in the presence of a condensation agent;

[0032]condensation of a compound (amino acid, peptide) with an activated
carboxyl group and free or protected other reaction groups with a
compound (amino acid, peptide) with a free amino group and free or
protected other reactive groups.

[0033]Activation of the carboxyl group can take place, inter alia, by
converting the carboxyl group to an acid halide, azide, anhydride,
imidazolide or an activated ester, such as the N-hydroxy-succinimide,
N-hydroxy-benzotriazole or p-nitrophenyl ester.

[0034]The most common methods for the above condensation reactions are:
the carbodiimide method, the azide method, the mixed anhydride method and
the method using activated esters, such as described in The Peptides,
Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.)
1979, 1980, 1981 (Academic Press, Inc.).

[0035]Production of peptides by recombinant DNA techniques is a general
method which is known, but which has a lot of possibilities all leading
to somewhat different results. The polypeptide to be expressed is coded
for by a DNA sequence or more accurately by a nucleic acid sequence.

[0036]Also part of the invention is the nucleic acid sequence comprising
the sequence encoding the peptides according to the present invention.

[0037]Preferably, the sequence encoding the peptides according to the
present invention are the sequences shown in SEQ ID NOS: 1-4 and 32-34.

[0038]As is well known in the art, the degeneracy of the genetic code
permits substitution of bases in a codon to result in another codon still
coding for the same amino acid, e.g., the codon for the amino acid
glutamic acid is both GAT and GAA. Consequently, it is clear that for the
expression of a polypeptide with an amino acid sequence as shown in SEQ
ID NO: 1-4, 9 or 32-34 use can be made of a derivate nucleic acid
sequence with such an alternative codon composition thereby different
nucleic acid sequences can be found.

[0039]"Nucleotide sequence" as used herein refers to a polymeric form of
nucleotides of any length, both to ribonucleic acid (RNA) sequences and
to deoxyribonucleic acid (DNA) sequences. In principle, this term refers
to the primary structure of the molecule. Thus, this term includes double
and single stranded DNA, as well as double and single stranded RNA, and
modifications thereof.

[0040]A further part of the invention are peptides, which are immunogenic
fragments of the peptide-analogues.

[0041]Immunogenic fragments are fragments which still have the ability to
induce an immunogenic response, i.e., that it is either possible to evoke
antibodies recognizing the fragments specifically, or that it is possible
to find T lymphocytes which have been activated by the fragments. Another
possibility is a DNA vaccine.

[0042]As has been said above, it has been known that the immunogenic
action of tumor associated antigens is often elicited through a T cell
activating mechanism (Townsend et al., 1989, H., Ann. Rev. Immunol. 7,
601-624). Cytotoxic T lymphocytes (CTLs) recognizing melanoma cells in a
T-cell receptor (TCR)-dependent and MHC-restricted manner have been
isolated from tumor-bearing patients (Knuth et al., 1992, Cancer surveys,
39-52). It has been shown that a peptide derived from tyrosinase, another
melanocyte-specific antigen, is recognized by a CTL clone (Brichard et
al., 1993, J. Exp. Med., 178, 489-495).

[0043]It is known that the activation of T cells through the MHC molecule
necessitates processing of the antigen of which short pieces (for example
8-12 mers) are presented to the T lymphocyte.

[0044]Preferably, the peptides according to the present invention are
flanked by non-related sequences, i.e., sequences with which they are not
connected in nature, because it has been found that such flanking
enhances the immunogenic properties of these peptides, probably through a
better processing and presentation by APCs.

[0045]Another part of the invention is formed by nucleotide sequences
comprising the nucleotide sequences coding for the above mentioned
peptides or an array of peptides.

[0046]Next to the use of these sequences for the production of the
peptides with recombinant DNA techniques, which will be exemplified
further, the sequence information disclosed in the sequence listings for
the peptides according to the present invention can be used for
diagnostic purposes.

[0047]From these sequences primers can be derived as basis for a
diagnostic test to detect gp100 or gp100-like proteins by a nucleic acid
amplification technique for instance the polymerase chain reaction (PCR)
or the nucleic acid sequence based amplification (NASBA) as described in
U.S. Pat. No. 4,683,202 and EP 329,822, respectively.

[0048]These nucleotide sequences can be used for the production of the
peptides according to the present invention with recombinant DNA
techniques. For this, the nucleotide sequence must be comprised in a
cloning vehicle which can be used to transform or transfect a suitable
host cell.

[0049]A wide variety of host cell and cloning vehicle combinations may be
usefully employed in cloning the nucleic acid sequence. For example,
useful cloning vehicles may include chromosomal, non-chromosomal and
synthetic DNA sequences such as various known bacterial plasmids, and
wider host range plasmids such as pBR 322, the various pUC, pGEM and
pBluescript plasmids, bacteriophages, e.g. lambda-gt-Wes, Charon 28 and
the M13 derived phages and vectors derived from combinations of plasmids
and phage or virus DNA, such as SV40, adenovirus or polyoma virus DNA
(Rodriquez et al., 1988, ed. Vectors, Butterworths; Lenstra et al., 1990,
Arch. Virol., 110, 1-24).

[0050]Useful hosts may include bacterial hosts, yeasts and other fungi,
plant or animal hosts, such as Chinese Hamster Ovary (CHO) cells,
melanoma cells, dendritic cells, monkey cells and other hosts.

[0051]Vehicles for use in expression of the peptides may further comprise
control sequences operably linked to the nucleic acid sequence coding for
the peptide. Such control sequences generally comprise a promoter
sequence and sequences which regulate and/or enhance expression levels.
Furthermore, an origin of replication and/or a dominant selection marker
are often present in such vehicles. Of course, control and other
sequences can vary depending on the host cell selected.

[0053]It is extremely practical if, next to the information for the
peptide, also the host cell is co-transformed or co-transfected with a
vector which carries the information for an MHC molecule to which said
peptide is known to bind. Preferably, the MHC molecule is HLA-A2.1,
HLA-A1 or HLA-A3.1, or any other HLA allele which is known to be present
in melanoma patients. HLA-A2.1 is especially preferred because it has
been established (Anichini et al., 1993, J. Exp. Med., 177, 989-998) that
melanoma cells carry antigens recognized by HLA-A2.1 restricted cytotoxic
T cell clones from melanoma patients.

[0054]Host cells especially suited for the expression of the peptides
according to the present invention are the murine EL4 and P8.15 cells.
For expression of said peptides human BLM cells (Katano et al., 1984, J.
Cancer Res. Clin. Oncol. 108, 197) are especially suited because they
already are able to express the MHC molecule HLA-A2.1.

[0055]The peptides according to the present invention can be used in a
vaccine for the treatment of melanoma.

[0056]In addition to an immunogenically effective amount of the active
peptide, the vaccine may contain a pharmaceutically acceptable carrier or
diluent.

[0057]The immunogenicity of the peptides of the invention, especially the
oligopeptides, can be enhanced by cross-linking or by coupling to an
immunogenic carrier molecule (i.e., a macromolecule having the property
of independently eliciting an immunological response in a patient, to
which the peptides of the invention can be covalently linked) or if part
of a protein.

[0058]Covalent coupling to the carrier molecule can be carried out using
methods well known in the art, the exact choice of which will be dictated
by the nature of the carrier molecule used. When the immunogenic carrier
molecule is a protein, the peptides of the invention can be coupled,
e.g., using water soluble carbodiimides such as dicyclohexylcarbodiimide,
or glutaraldehyde.

[0059]Coupling agents such as these can also be used to cross-link the
peptides to themselves without the use of a separate carrier molecule.
Such cross-linking into polypeptides or peptide aggregates can also
increase immunogenicity.

[0061]Optionally, one or more compounds having adjuvant activity may be
added to the vaccine. Suitable adjuvants are for example aluminium
hydroxide, phosphate or oxide, oil-emulsions (e.g. of Bayol F® or
Marcol 52®), saponins or vitamin-E solubilisate.

[0062]Dendritic cells are professional APC that express mannose receptor
used to take up antigen thus facilitating antigen processing.

[0063]The vaccine according to the present invention can be given inter
alia intravenously, intraperitoneally, intranasally, intradermally,
subcutaneously or intramuscularly.

[0064]The useful effective amount to be administered will vary depending
on the age and weight of the patient and mode of administration of the
vaccine.

[0065]The vaccine can be employed to specifically obtain a T cell
response, but it is also possible that a B cell response is elicited
after vaccination. If so, the B cell response leads to the formation of
antibodies against the peptide of the vaccine, which antibodies will be
directed to the source of the antigen production, i.e., the tumor cells.
This is an advantageous feature, because in this way the tumor cells are
combated by responses of both the immunological systems.

[0066]Both immunological systems will even be more effectively triggered
when the vaccine comprises the peptides as presented in an MHC molecule
by an antigen presenting cell (APC). Antigen presentation can be achieved
by using monocytes, macrophages, interdigitating cells, Langerhans cells
and especially dendritic cells, loaded with one of the peptides of the
invention or loading with protein including peptide or manosylated
protein. Loading of the APCs can be accomplished by bringing the peptides
of the invention into or in the neighborhood of the APC, but it is more
preferable to let the APC process the complete gp100 antigen. In this way
a presentation is achieved which mimics the in vivo situation most
realistically. Furthermore, the MHC used by the cell is of the type which
is suited to present the epitope.

[0067]An overall advantage of using APCs for the presentation of the
epitopes is the choice of APC cell that is used in this respect. It is
known from different types of APCs that there are stimulating APCs and
inhibiting APCs.

[0068]Preferred APCs include, but are not limited to, the listed cell
types, which are so-called "professional" antigen presenting cells,
characterized in that they have co-stimulating molecules, which have an
important function in the process of antigen presentation. Such
co-stimulating molecules are, for example, B7, CD25, CD40, CD70, CTLA-4
or heat stable antigen (Schwartz, 1992, Cell 71, 1065-1068).

[0069]Fibroblasts, which have also been shown to be able to act as an
antigen presenting cell, lack these co-stimulating molecules.

[0070]It is also possible to use cells already transfected with a cloning
vehicle harboring the information for the melanocyte peptide analogues
and which are cotransfected with a cloning vehicle which comprises the
nucleotide sequence for an MHC class I molecule, for instance the
sequence coding for HLA A2.1, HLA A1 or HLA A3.1. These cells will act as
an antigen presenting cell and will present peptide analogues in the MHC
class I molecules which are expressed on their surface. It is envisaged
that this presentation will be enhanced, when the cell is also capable of
expressing one of the above-mentioned co-stimulating molecules (in
particular B7 (B7.1, B7.2), CD40), or a molecule with a similar function
(e.g., cytokines transfected in cell line). This expression can be the
result of transformation or transfection of the cell with a third cloning
vehicle having the sequence information coding for such a co-stimulating
molecule, but it can also be that the cell already was capable of
production of co-stimulating molecules.

[0071]Instead of a vaccine with these cells, which next to the desired
expression products, also harbor many elements which are also expressed
and which can negatively affect the desired immunogenic reaction of the
cell, it is also possible that a vaccine is composed with liposomes which
expose MHC molecules loaded with peptides, and which, for instance, are
filled with lymphokines. Such liposomes will trigger an immunologic T
cell reaction.

[0072]By presenting the peptide in the same way as it is also presented in
vivo, an enhanced T cell response will be evoked. Furthermore, by the
natural adjuvant working of the relatively large, antigen presenting
cells also a B cell response is triggered. This B cell response will also
lead to the formation of antibodies directed to the peptide-MHC complex.
This complex is especially found in tumor cells, where it has been shown
that in the patient epitopes of gp100 are presented naturally, which are
thus able to elicit a T cell response. It is this naturally occurring
phenomenon which is enlarged by the vaccination of APCs already
presenting the peptides of the invention. By enlarging not only an
enlarged T cell response will be evoked, but also a B cell response which
leads to antibodies directed to the MHC-peptide complex will be
initiated.

[0073]The vaccines according to the invention can be enriched by numerous
compounds which have an enhancing effect on the initiation and the
maintenance of both the T cell and the B cell response after vaccination.

[0074]In this way, addition of cytokines to the vaccine will enhance the T
cell response. Suitable cytokines are for instance interleukins, such as
IL-2, IL-4, IL-7, or IL-12, GM-CSF, RANTES, MIP-α, and tumor
necrosis factor, and interferons, such as IFN- or the chemokins.

[0075]In a similar way, antibodies against T cell surface antigens, such
as CD2, CD3, CD27 and CD28 will enhance the immunogenic reaction.

[0076]Also, the addition of helper epitopes to stimulate CD4.sup.+ helper
cells or CD8.sup.+ killer cells augments the immunogenic reaction.
Alternatively, also helper epitopes from other antigens can be used, for
instance from heat shock derived proteins or cholera toxin.

[0077]Another part of the invention is formed by using reactive tumor
infiltrating lymphocytes (TILs) directed against the peptides according
to the present invention. In this method, the first step is taking a
sample from a patient. This is usually done by resection of a tumor
deposit under local anesthesia. The TILs present in this specimen are
then expanded in culture for four to eight weeks, according to known
methods (Topalian et al., 1987, J. Immunol. Meth. 102, 127-141). During
this culture, the TILs are then checked for reactivity with the peptides
according to the present invention or gp100-protein. The TILs which
recognize the antigen are isolated and cultured further.

[0078]The reactive tumor infiltrating lymphocytes which are obtained
through this method, also form part of the invention. An example of such
TIL cell line, designated TIL 1200, has been found which specifically
reacts with gp100 and its epitopes. This TIL 1200 cell line also
expresses the MHC molecule HLA-A2.1. Furthermore, expression of TCR
α/β, CD3 and CD8 by this cell line has been demonstrated.
Furthermore, TIL 1200 recognizes transfectants expressing both HLA-A2.1
and gp100 .

[0079]TIL 1200 and other TILs recognizing gp100 are suited for treatment
of melanoma patients. For such treatment, TILs may be cultured as stated
above, and they are given back to the patients by an intravenous
infusion. The success of treatment can be enhanced by pre-treatment of
the tumor bearing host with either total body radiation or treatment with
cyclophosphamide and by the simultaneous administration of interleukin-2
(Rosenberg et al., 1986, Science 223, 1318-1321).

[0080]The TILs infused back to the patient are preferably autologous TILs
(i.e., derived from the patient's own tumor) but also infusion with
allogenic TILs can be imagined.

[0081]A further use of the TILs obtained by the method as described above
is for in vivo diagnosis. Labeling of the TILs, for instance with
111In (Fisher et al., 1989, J. Clin. Oncol. 7, 250-261) or any other
suitable diagnostic marker, renders them suited for identification of
tumor deposits in melanoma patients.

[0082]Another part of the invention is formed by the T cell receptor (TCR)
expressed by reactive CTLs directed against the peptides according to
this invention or the gp100-protein. As is well known in the art, the TCR
determines the specificity of a CTL. Therefore, the cDNA encoding the
TCR, especially its variable region, can be isolated and introduced into
T cells, thereby transferring anti-tumor activity to any T cell.
Especially introduction of such a TCR into autologous T cells and
subsequent expansion of these T cells will result in large numbers of CTL
suitable for adoptive transfer into the autologous patient.

[0083]Cells harboring this T cell receptor can also be used for
vaccination purposes.

[0084]A vaccine can also be composed from melanoma cells capable of
expression of the peptides according to the present invention. It is
possible to isolate these cells from a patient, using specific
antibodies, such as NKI-beteb (directed against gp100), but is also
possible to produce such melanoma cells from cultured melanoma cell
lines, which either are natural gp100-producers or have been manipulated
genetically to produce the peptides according to the present invention.
These cells can be irradiated to be non-tumorogenic and infused (back)
into the patient. To enhance the immunologic effect of these melanoma
cells it is preferred to alter them genetically to produce a lymphokine,
preferably interleukine-2 (IL-2) or granulocyte-macrophage colony
stimulation factor (GM-CSF). Peptide.sup.+/gp100.sup.+ melanoma cells can
be transfected with a cloning vehicle having the sequence coding for the
production of IL-2 or GM-CSF.

[0085]Infusion of such a vaccine into a patient will stimulate the
formation of CTLs.

[0086]Another type of vaccination having a similar effect is vaccination
with pure DNA, for instance the DNA of a vector or a vector virus having
the DNA sequence encoding the peptides according the present invention
(both homologues and heterologues (chimeric protein) or repetitive). Once
injected, the virus will infect or the DNA will be transformed to cells
which express the antigen or the peptide(s).

[0087]Antibodies directed against the peptides according to the present
invention are also part of the invention.

[0088]Monospecific antibodies to these peptides can be obtained by
affinity purification from polyspecific antisera by a modification of the
method of Hall et al. (1984, Nature 311, 379-387). Polyspecific antisera
can be obtained by immunizing rabbits according to standard immunization
schemes.

[0089]Monospecific antibody as used herein is defined as a single antibody
species or multiple antibody species with homogeneous binding
characteristics for the relevant antigen. Homogeneous binding as used
herein refers to the ability of the antibody species to bind to ligand
binding domain of the invention.

[0090]The antibody is preferably a monoclonal antibody, more preferably a
humanized monoclonal antibody.

[0091]Monoclonal antibodies can be prepared by immunizing inbred nice,
preferably Balb/c with the appropriate protein by techniques known in the
art (Kohler, G. and Milstein C., 1975, Nature 256, 495-497). Hybridoma
cells are subsequently selected by growth in hypoxanthine, thymidine and
aminopterin in an appropriate cell culture medium such as Dulbecco's
modified Eagle's medium (DMEM). Antibody producing hybridomas are cloned,
preferably using the soft agar technique of MacPherson (1973, Tissue
Culture Methods and Applications, Kruse and Paterson, eds., Academic
Press). Discrete colonies are transferred into individual wells of
culture plates for cultivation in an appropriate culture medium. Antibody
producing cells are identified by screening with the appropriate
immunogen. Immunogen positive hybridoma cells are maintained by
techniques known in the art. Specific anti-monoclonal antibodies are
produced by cultivating the hybridomas in vitro or preparing ascites
fluid in mice following hybridoma injection by procedures known in the
art.

[0092]It may be preferred to use humanized antibodies. Methods for
humanizing antibodies, such as CDR-grafting, are known (Jones et al.,
1986, Nature 321, 522-525). Another possibility to avoid antigenic
response to antibodies reactive with polypeptides according to the
invention is the use of human antibodies or fragments or derivatives
thereof.

[0093]Human antibodies can be produced by in vitro stimulation of isolated
B-lymphocytes, or they can be isolated from (immortalized) B-lymphocytes
which have been harvested from a human being immunized with at least one
ligand binding domain according to the invention.

[0094]Antibodies as described above can be used for the passive
vaccination of melanoma patients. A preferred type of antibodies for this
kind of vaccine are antibodies directed against the above-mentioned
peptides presented in connection with the MHC molecule. To produce these
kind of antibodies immunization of peptides presented by APCs is
required. Such an immunization can be performed as described above.
Alternatively, human antibodies to peptide-MHC complexes can be isolated
from patients treated with a vaccine consisting of APCs loaded with one
of said peptides.

[0095]The antibodies, which are formed after treatment with one of the
vaccines of the invention can also be used for the monitoring of said
vaccination. For such a method, serum of the patients is obtained and the
antibodies directed to the peptide with which has been vaccinated are
detected. Knowing the antibody titre from this detection, it can be
judged if there is need for a boost vaccination.

[0096]Specific detection of said antibodies in the serum can be achieved
by labeled peptides. The label can be any diagnostic marker known in the
field of in vitro diagnosis, but most preferred (and widely used) are
enzymes, dyes, metals and radionuclides, such as 67Ga, 99mTC,
111In, .sup.113mIn, 123I, 125I, or 131I.

[0097]The radiodiagnostic markers can be coupled directly to the peptides
of the invention or through chelating moieties which have been coupled to
the peptide directly or through linker or spacer molecules. The technique
of coupling of radionuclides to peptides or peptide-like structures is
already known in the field of (tumor) diagnostics from the numerous
applications of labeled antibodies used both in in vivo and in in vitro
tests.

[0098]Direct labeling of peptides can, for instance, be performed as
described in the one-vial method (Haisma et al., 1986, J. Nucl. Med. 27,
1890). A general method for labeling of peptides through chelators, with
or without linker or spacer molecules, has, for instance, been described
in U.S. Pat. Nos. 4,472,509 and 4,485,086. Chelators using a bicyclic
anhydride of DTPA have been disclosed in Hnatowich et al. (1983, J.
Immunol. Meth. 65, 147-157). Coupling through diamide dimercaptide
compounds has been disclosed in EP 188,256.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0099]The present invention is further described by way of examples with
reference to the accompanying figures, in which:

[0102]FIG. 3. Immunogenicity of gp100 154-162 epitope-analogues in
HLA-A*0201/Kb transgenic mice. Bulk CTL obtained from immunized mice
were tested for lytic activity using chromium labeled Jurkat A2/Kb
target cells that were preincubated with no peptide, 10 mM wild type
gp100 154-162 or 10 mM of the epitope-analogue used to immunize the mice.
For each peptide the mean specific lysis of bulk CTL of the responding
mice is shown. Standard deviations never exceeded 15% of the mean value.
One representative experiment out of two is shown.

[0103]FIG. 4. Peptide specific reactivity of in vitro induced
epitope-analogue specific CTL cultures. Chromium-labeled HLA-A*0201.sup.+
T2 target cells were pre-incubated with 10 mM of an irrelevant
HLA-A*0201-binding peptide, 10 mM wild type gp100 154-162 or 10 mM of the
epitope-analogue used for CTL induction. The different CTL cultures were
added at an effector to target ratio of 20:1. One representative
experiment out of two is shown.

[0104]FIG. 5. Epitope-analogue induced CTL cultures specifically lyse
melanoma cells endogenously presenting the wild type epitope.
Chromium-labeled HLA-A2.1.sup.+ BLM and Mel 624 melanoma cells were used
as target cells. BLM cells lack expression of gp100. The different CTL
cultures were added at an effector to target ratio of 20:1. One
representative experiment out of two is shown.

[0107]HLA-A*0201/Kb transgenic mice were used (animal distributor
Harlan Sprague Dawley, Inc., Indianapolis, USA). Mice were held under
clean conventional conditions. The transgenic mice express the product of
the HLA-A*0201/Kb chimeric gene in which the α3 domain of the
heavy chain is replaced by the corresponding murine H-2 Kb domain
while leaving the HLA-A*0201 α1 and α2 domains unaffected
(Vitiello et al., 1991, J. Exp. Med. 1007). This allows the murine CD8
molecule on the murine CD8.sup.+ T lymphocytes to interact with the
syngeneic α3 domain of the hybrid MHC class I molecule.

Peptides.

[0108]For induction of CTL and chromium-release assays, peptides were
synthesized with a free carboxy-terminus by Fmoc peptide chemistry using
an ABIMED multiple synthesizer. All peptides were >90% pure as
indicated by analytical HPLC. Peptides were dissolved in DMSO and stored
at -20° C.

[0110]Peptide-binding to HLA-A*0201 was analyzed using HLA-A*0201.sup.+ JY
cells as was described previously (van der Burg et al., 1995, Hum.
Immunol. 44:189). Briefly, mild-acid treated JY cells were incubated with
150 nM Fluorescein (FL)-labeled reference peptide (FLPSDC(-FL)FPSV) and
with several concentrations of competitor peptide for 24 hours at
4° in the presence of 1.0 mg/ml β2-microglobulin (Sigma, St.
Louis, Mo.). Subsequently, the cells were washed, fixed with
paraformaldehyde and analyzed by flow cytometry. The mean-fluorescence
(MF) obtained in the absence of competitor peptide was regarded as
maximal binding and equated to 0%; the MF obtained without reference
peptide was equated to 100% inhibition. % inhibition of binding was
calculated using the formula: (1-(MF 150 nM reference & competitor
peptide-MF no reference peptide)/(MF 150 nM reference peptide-MF no
reference peptide))×100%. The binding capacity of competitor
peptides is expressed as the concentration needed to inhibit 50% of
binding of the FL-labeled reference peptide (IC50).

Measurement of MHC-Peptide Complex Stability at 37° C.

[0111]Measurement of MHC-peptide complex stability was performed.
HLA-A*0201.sup.+ homozygous JY cells were treated with 10-4 M
emetine (Sigma, St. Louis, USA) for 1 hour at 37° C. to stop de
novo synthesis of MHC class I molecules. The cells were then mild-acid
treated and subsequently loaded with 200 mM of peptide for 1 hour at room
temperature. Thereafter, the cells were washed twice to remove free
peptide and were incubated at 37° C. for 0, 2, 4 and 6 hours.
Subsequently, the cells were stained using mAb BB7.2 (Parham et al.,
1981, Hum. Immunol. 3:277), fixed with paraformaldehyde and analyzed by
flow cytometry.

[0114]On day 8 and day 15, the responder populations were restimulated
using peptide-pulsed dendritic cells as stimulator cells. The cultures
were propagated in medium containing IL-2 (Cetus Corp., Emeryville,
Calif.) and IL-7 (Genzyme, Cambridge, Mass.) at final concentrations of
10 U/ml and 5 ng/ml respectively. Weekly hereafter the cultures were
restimulated using adherent peptide-pulsed PBMC as was described
previously (Bakker et al., 1995, Cancer Res. 55:5330). Responder
populations were tested for specific lytic activity after at least 4
rounds of restimulation.

Chromium-Release Assay.

[0115]Chromium release assays were performed as described previously
(Bakker et al., 1994, J. Exp. Med. 179:1005.). Briefly, 106 target
cells were incubated with 100 mCi Na251CrO4 (Amersham,
Bucks, UK) for 1 hour. Various amounts of effector cells were then added
to the target cells in triplicate wells of U bottomed microtiter plates
(Costar, Badhoevedorp, The Netherlands) in a final volume of 150 ml. In
peptide recognition assays, target cells were pre-incubated with various
concentrations of peptide for 30 or 60 min at 37° C. in a volume
of 100 ml prior to the addition of effector cells. After 5 h of
incubation, part of the supernatant was harvested and its radioactive
content was measured. The mean percentage specific lysis of triplicate
wells was calculated using the formula: % specific lysis=((experimental
release-spontaneous release)/(maximal release-spontaneous
release))×100.

[0116]The Melan-A/MART-27-35 and the gp100 154-162 epitopes have been
identified using HLA-A*0201 restricted TIL lines derived from metastatic
melanomas. The Melan-A/MART-1 27-35 epitope was found to be the nominal
epitope capable of triggering the Melan-A/MART-1 specific TIL 1235 line
when presented on HLA-A*0201.sup.+ target cells (Kawakami et al., 1994.
J. Exp. Med. 180:347). Among a panel of peptides ranging from 8-mers to
11-mers located around gp100 amino acids 155-161, we identified the 9-mer
154-162 as the peptide most efficient in sensitizing HLA-A*0201.sup.+
target cells for lysis by the gp100 reactive TIL 1200 line (Balder et
al., 1995, Int. J. Cancer 62:97). Both the Melan-A/MART-1 27-35 9-mer and
the gp100 154-162 9-mer have now been eluted from the cell surface of
HLA-A*0201.sup.+ melanoma cells, and were identified by tandem
mass-spectroscopy, indicating that they are indeed the nominal epitopes
endogenously presented in HLA-A*0201. To identify amino acid residues in
both epitopes engaged in HLA-A*0201 binding and/or TCR interactions,
epitope-analogues were synthesized in which the native amino acid was
replaced by an alanine residue. In case alanine residues were present in
the wild type epitope, they were substituted for the amino acid glycine.
The substituted peptides were assayed for binding to HLA-A*0201 by means
of an indirect binding assay using the processing defective cell line T2
(Nijman et al., 1993, Eur. J. Immunol. 23:1215). All substitutions in the
Melan-A/MART-1 epitope resulted in a nearly complete loss in the
capability to stabilize HLA-A*0201 molecules at the cell surface of T2
cells (Table I). When the Melan-A/MART-1 27-35 analogues were used at
micromolar concentrations to sensitize HLA-A*0201.sup.+ target cells for
lysis by Melan-A/MART-1-specific CTL, we observed a decrease in target
cell lysis for the alanine replacements at positions 4 to 7 of the
epitope (Table I). In addition, the glycine substitution at position 2
resulted in decreased CTL reactivity. The amino acids at these positions
in the Melan-A/MART-1 27-35 epitope are therefore most likely involved in
TCR interactions.

[0117]In case of the gp100 154-162 epitope decreased HLA-A*0201 affinity
of epitope-analogues was only observed for the alanine substitutions at
position 3 and 9 (Table 1). With respect to T cell recognition, alanine
substitutions at positions 5, 6 and 7 of the epitope were not allowed,
indicating that amino acids at these positions are critical contact
residues within this epitope for the TCR.

[0118]Subsequently, the epitope-analogues that induced reactivity at
micromolar concentrations were titrated to evaluate their relative
ability to sensitize T2 target cells for lysis by the relevant CTL (FIG.
1). In all cases the epitope-analogues were similar or inferior compared
to the wild type epitope in their sensitizing capacity, except for the
alanine substitution at position 8 of the gp100 154-162 epitope.
Surprisingly, this peptide was able to induce target cell lysis by
gp100-reactive CTL even at a tenfold lower concentration.

Example 2

N-Terminal Anchor Residue Replacements in Both the gp100 154-162 and the
Melan-A/MART-1 27-35 Epitopes Result in Improved Affinity for HLA-A*0201

[0119]Since both the Melan-A/MART-1 27-35 and the gp100 154-162 epitopes
have non-conventional N-terminal anchoring residues, we replaced these
residues for the common HLA-A*0201 anchoring residues V, L, I or M
(Drijfhout et al., 1995, Hum. Immunol. 43:1). Subsequently, we tested
these peptides for HLA-A*0201 binding and their ability to sensitize
target cells for lysis by the relevant CTL. Apart from the methionine
substitution, all anchor residue replacements in the Melan-A/MART-1
epitope resulted in significantly improved binding to HLA-A*0201 (Table
II). HLA-A*0201.sup.+ target cells loaded with these peptides at a
concentration of 1 mM were recognized by the Melan-A/MART-1 reactive CTL,
except for the methionine substituted epitope (Table II). Although this
peptide did bind to HLA-A*0201 at a level comparable to the wild type
epitope, it failed to induce CTL reactivity. Titration experiments using
the Melan-A/MART-1 anchor replacement peptides demonstrated that these
epitope-analogues were inferior to wild type in sensitizing target cells
for lysis by TIL 1235 (FIG. 2).

[0121]To assess whether the augmented CTL recognition of the substituted
gp100 154-162 epitopes could be attributed to improved HLA-A*0201
affinity, the HLA-A*0201 binding capacity of these peptides was tested
now using a more sensitive cell-bound HLA-A*0201 binding assay based on
competition of a labeled reference peptide with the peptides of interest
(van der Burg et al., 1995, Hum. Immunol. 44:189). HLA-A*0201
binding-affinities obtained with this assay demonstrated that all
peptides that were able to sensitize target cells for lysis by TIL 1200
at tenfold lower concentrations compared to wild type, also bound with
higher affinity to HLA-A*0201 (Table III). In addition to the N-terminal
anchor substitutions, replacement of a polar residue for a hydrophobic
residue adjacent to the C-terminal anchoring position also resulted in an
epitope-analogue with improved HLA-A*0201 affinity (KTWGQYWAV (SEQ ID NO:
1)), apparently without affecting TCR recognition. Measurement of MHC
class I-peptide complex dissociation rates demonstrated that the
epitope-analogues tested are at least equally stable when compared to
wild type (Table III). All peptides tested showed a DT50 (the time
required for 50% of the complexes to decay) longer than 4 hours. Peptides
with DT50 values of ≧3 hours were immunogenic in
HLA-A*0201/Kb transgenic mice. Taken together, these data indicate
that the gp100 154-162 epitope-analogues may have similar or increased
immunogenicity compared to wild type gp100 154-162.